A central goal of evolutionary biology is to elucidate the genetic architecture of adaptation. In humans, this question is of interest both for what it will reveal about our species-specific traits and because of the emerging links between adaptation and disease susceptibility. To date, however, there are only a handful of examples of human regulatory adaptations, such that many outstanding questions remain open. Among these: Which pathways have been remodeled in human evolution? Do adaptive changes in the regulation of entire pathways involve changes to many genes, or to few? What is the relative importance of changes in cis (e.g., promoter regions) vs. trans (e.g., transcription factors) regulatory elements? How prevalent are compensatory changes in regulatory pathways? As a first step towards answering these questions, we propose to identify transcriptional pathways that have been adaptively remodeled in humans and to examine their evolution across three primate species. Specifically, we plan to focus on five transcription factors that have been shown previously (in our work and by others) to be the target of positive selection in the human lineage. Through a combination of siRNA knockdowns, gene expression profiles, ChIP-seq, and reporter gene experiments, we will identify the genes that are directly regulated by these transcription factors, not only in humans but also in two close evolutionary relatives, chimpanzees and rhesus macaques. The proposed combination of approaches will lead to the reliable annotation of direct regulatory targets of five transcription factors in three species and facilitate the identification of transcriptional pathways that underlie human-specific adaptation. Comparison of regulatory networks in the three species will reveal the genetic basis for a large set of regulatory differences between humans and closely related species, enabling us to address many of the above questions. To our knowledge, this research represents the first genome-wide exploration of differences in regulatory pathways across species. In addition to identifying pathways that have been adaptively remodeled in the human lineage, it will yield unprecedented insights into the genetic basis of regulatory change at the transcription level. PUBLIC HEALTH RELEVANCE: The goal of the proposed study is to identify a first set of regulatory pathways that have been remodeled in humans, and learn about the genetic basis of gene regulatory changes in primates.